In scanning microscopy, a specimen is illuminated with a light beam in order to observe the detected light, constituting reflected or fluorescent light, emitted by the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable scanning device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. The scanning device is triggered with a nominal signal. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements; both fast resonant as well as slower (more accurate) non-resonant galvanometers are used. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to determine the present mirror position (actual signal). The actual signal is usually assigned unequivocally to the respective detection signal so that an image can be generated.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture stop (called the “excitation stop”), a beam splitter, a scanning device for beam control, a microscope optical system, a detection stop, and the detectors for detecting the detection or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen arrives by way of the scanning device back at the beam splitter, passes through the latter, and is then focused onto the detection stop behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop, so that a point datum is obtained which results, by successive scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers. Commercial scanning microscopes usually comprise a scanning module that is flange-mounted onto the stand of a conventional light microscope, the scanning module additionally containing all the aforesaid elements necessary for scanning a specimen.
In confocal scanning microscopy, a detection stop can be dispensed with in the case of two-photon (or multi-photon) excitation, since the excitation probability depends on the square of the photon density and thus on the square of the illuminating light intensity, which of course is much greater at the focus than in the adjacent regions. The fluorescent light being detected therefore very probably originates almost exclusively from the focus region, which renders superfluous any further differentiation, using a stop arrangement, between fluorescent photons from the focus region and fluorescent photons from the adjacent regions.
Ideally, the track of the scanning light beam on or in the specimen should describe a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). In reality, however, this is not achieved as a result of various interference effects, so that troublesome image defects occur. In particular, the inertia of the moving components, positioning elements, and mirrors permits a meander-shaped scanning path only when scanning very slowly. With rapid scanning, the positioning elements are preferably triggered in sawtooth form (linearly over time), or sinusoidally (almost linearly in the central region). In actuality, the positioning elements do not exactly follow the nominal signal when scanning rapidly. The scanning track of the light beam describes a sine-like curve in the specimen. A further error source may be found in the fact that the projection of the path speed onto the X direction is less in the vicinity of the reversal points than in the linear region of the sine-like curve. Often, for example with “bad” galvanometers, an enormous deviation from the sine shape is in fact evident. It also happens that the curve shape for deflection in the positive X direction differs from the curve shape upon deflection in the opposite, negative X direction.
Image defects also occur because of inaccuracies in the sensors for determining the present mirror position, i.e. because of errors in the actual signal. These sensor measurement errors are principally attributable to friction and magnetization of the material.
DE Unexamined Application 197 02 752 A1 discloses a triggering system for a scanner drive, in particular for a laser scanning microscope, having an oscillating motor for driving an oscillating mirror that serves for linearly oscillating deflection of a beam; having a triggering unit for supplying to the oscillating motor an energizing current that is modifiable in terms of triggering frequency, frequency curve, and amplitude; having a function generator that is connected to the triggering unit; and having a measured value transducer for obtaining a sequence of data concerning the deflection positions of the oscillating mirror. The object of the invention is achieved in that the measured value transducer is linked to the function generator via a logic unit for determining correction values for the energizing current. It is thereby advantageously possible to determine correction values by means of the logic unit by evaluating the data made available by the measured value transducer regarding the actual deflection position of the oscillating mirror. Those values can in turn be used to influence the triggering frequencies output by the function generator in such a way that the deviations are minimized or completely eliminated. The manner in which the stated problem is solved by the disclosed regulated triggering system for the positioning elements is technically very complex and expensive. The disclosed triggering system is moreover limited, since at very high scanning speeds a nonlinear scanning track always at least partially occurs. When the positioning elements are resonantly operating galvanometers, practically only sine-like scanning tracks can be achieved.